ABSTRACT The tumor suppressor gene TP53 is the most commonly mutated gene in human cancers. Understanding the full breadth of p53 function and regulation is crucial to our understanding of tumor initiation and development. The MDM2 proto-oncoprotein is a primary negative regulator for p53 by promoting p53 polyubiquitination and proteosomal degradation. The MDM2 homologous protein MDMX also functions as a negative regulator for p53. While many questions remain, a focus on the use of in vivo models is allowing for a closer view of how this MDM2/MDMX-p53 pathway is regulated, with a newfound emphasis on how this pathway can be better manipulated for therapeutic gains. During the last few years, our experiments have been focused on generating and examining MDM2 mutation knock-in mice that are deficient in MDM2 E3 ligase function or MDM2-MDMX binding. We have generated MDM2C462A and MDM2Y487A mutant mice, both lacking MDM2 E3 ligase function. Studies with the MDM2C462A mice, which also lack MDM2-MDMX interaction, and the MDM2Y487A mice, which retain MDM2- MDMX interaction, have generated new insight into the in vivo regulation of p53 by MDM2 E3 ligase and the MDM2-MDMX heterooligomer. Recently, we have generated a number of new mouse models expressing an inducible p53 under various MDM2 and MDMX backgrounds. Our preliminary data generated with these inducible p53 mice have revealed several surprising findings. We found that MDMX can suppress p53 expression independent of MDM2 E3 ligase function; we found that in vivo MDMX is required for MDM2 mediated p53 degradation; and we found that the MDM2C462A mutant has gained a neomorphic function for stimulating p53 transcriptional activity. Experiments proposed in this application are designed to study (1) whether MDMX regulates p53 expression through modulating its translation; (2) what is the mechanism by which MDMX impacts on MDM2 mediated p53 degradation; (3) how the RING finger mutant MDM2 gained a function to activate p53. These experiments provide proof-of-principle evidence for exploring inhibition of MDM2 RING E3 ligase function and disruption of MDM2-MDMX interaction as potential drug development strategies targeting cancers expressing high levels of MDM2 and MDMX and WT p53.